运动上调海马蛋白酶体活性对神经发生的影响及其机制

Neurogenesis is the process in which the neurons newly derived from neural stem cells (NSCs) functionally integrate into the existing circuits. During ageing, NSCs progressively lose their proliferating and differentiating potentials, subsequently resulting in neurogenesis suppression that has been regarded as the critical factor contributive to neurodegenerative diseases. Evidence is accumulating to suggest an association between the proteasome activity and the stemness of NSCs. We have previously demonstrated that enhancement of proteasome activity could help maintain the normal viability of NSCs. Additionally, the recent data provided by us showed that proteasomes might be activated by physical exercise, concurrent with improved learning and memory functions. In the present study, we shall further unmask the role of proteasomes in modulating adult neurogenesis, including proliferation, differentiation, migration and integration of NSCs. Particularly, we shall in depth explore whether and how up-regulation of proteasome activity by persistent physical exercise could exert preventive and curative effects on mitigating cognitive deficits related to ageing, by use of the VitalView on-line wheel running system in combination with morphological, electrophysiological and behavioral methods. Furthermore, the high-throughput sequencing, siRNA library screening and high-content imaging will be applied to identify the key target genes that control the proteasome activity and thus affect the biological properties of NSCs. The overall goal of our study is to elucidate the mechanism mediating the physical exercise-elicited promotion of proteasome activity and neurogenesis; this will not only assist in devising new strategies to counteract ageing-exerted adverse effects on NSCs, but also set a theoretical basis for better advocating the active healthy lifestyle.

神经干细胞（NSCs）分化产生神经元，参与神经功能活动的过程称为神经发生。然而，随年龄增加NSCs增殖和分化能力降低，出现神经发生障碍，这是神经退行性疾病发生的关键事件。有证据显示蛋白酶体活性与干细胞“干性”相关。我们已证实上调蛋白酶体活性有助于维持NSCs活力，近期结果提示运动可增强海马蛋白酶体活性，提高小鼠认知功能，推测蛋白酶体活性是神经发生的重要环节。为此，本课题应用VitalView跑轮监测系统，在体研究运动上调成年和中老年鼠蛋白酶体活性对神经发生的影响。同时，采用形态学、电生理学和行为学手段，检测长期运动改善蛋白酶体活性对预防和治疗老年认知功能障碍的意义。最后，利用高通量测序和siRNA文库筛选平台，经高内涵成像系统挖掘运动激活蛋白酶体活性的关键靶点，分析靶基因对NSCs潜能的影响。旨在发现调控NSCs活力的新途径，全面解析运动促进神经发生的机制，为倡导健康生活方式提供理论依据。

运动有助于提高学习和记忆能力，改善年龄相关的认知功能障碍，降低神经退行性疾病的发生。然而，运动提高认知功能的机制尚未阐明。本研究首次证明随年龄增加海马蛋白酶体活性下降，自主跑轮运动能够显著增加成年鼠和中老年鼠海马蛋白酶体活性，提高神经发生相关标志物Ki67和DCX表达水平。相反，双侧海马注射蛋白酶体抑制剂MG132，能够降低运动对海马神经发生的促进作用。进一步研究证明，小鼠运动2周后血浆和海马中胰岛素样生长因子-1(IGF-1)表达水平升高。应用IGF-1直接作用于体外分离培养的成年神经前体细胞(aNPCs)，能够提高aNPCs的增殖和分化潜能，更为重要的是还能够提高aNPCs蛋白酶体活性，并且呈现剂量依赖性。转录因子E2相关因子2(Nrf2)作为IGF-1下游分子，已证明与细胞活力维持密切相关。本次研究中证明运动能够激活海马Nrf2转录水平，细胞核中Nrf2表达水平较对照组增加1.34倍（P<0.05）。应用100ng/mlIGF-1直接作用于aNPCs同样能够使细胞核中Nrf2表达水平增加1.22倍（P<0.05），并提高蛋白酶体核心亚单位PSMB5 mRNA的转录水平，提示IGF-1/Nrf2通路可能参与调控aNPCs蛋白酶体活性。腹腔注射IGF-1受体抑制剂picropodophyllin可降低运动小鼠海马Nrf2核转位，使蛋白酶体活性下降29.2%（P<0.05），同时免疫荧光染色结果表明海马Ki67和DCX阳性细胞数分别下降31.3%和34.9%（P<0.05），行为学实验结果表明Y迷宫自发交替率下降12%（P<0.05），新物体识别率下降13.2%（P<0.05），表明运动诱导的认知功能提高依赖于IGF-1诱导的蛋白酶体活性增加。双荧光素酶报告系统也证明，IGF-1可以增加Nrf2对PSMB5的转录激活作用，应用shRNA降低双侧海马Nrf2表达水平，运动后小鼠海马蛋白酶体活性较对照组下降33%（P<0.05），并且shRNA组小鼠的认知能力和工作记忆能力也降低。本项目通过体内和体外研究相结合的手段，揭示运动可能通过激活IGF-1/Nrf2信号通路上调海马蛋白酶体，进而促进神经发生，从而为预防和治疗神经退行性病变提供了新靶标。